Antibiotic substance

ABSTRACT

A novel antibiotic substance, named &#34;Mycoplanecin&#34;, of presently unknown structural formula, is produced by cultivating a Mycoplanecin-producing microorganism of the genus Actinoplanes, especially the Actinoplanes nov. sp. Strain No. 41042, NRRL No. 11462.

This is a division of application Ser. No. 41,501 filed May 22, 1979.

BACKGROUND AND SUMMARY OF THE INVENTION

The present invention relates to a novel antibiotic substance, named Mycoplanecin, to a novel microorganism, Actinoplanes nov. sp. Strain No. 41042 capable of producing Mycoplanecin, and to a process for producing Mycoplanecin by cultivating a Mycoplanecin-producing microorganism of the genus Actinoplanes.

The novel antibiotic substance, Mycoplanecin, can be characterized by the following physical and chemical properties:

1. Colour and state

White powder.

2. Melting point

161°-167° C.

3. Specific rotation

[α]_(D) ²¹ 66° (C=0.4, chloroform).

4. Elemental analysis

C, 61.78%; H, 8.48%; N, 11.75%. (After drying to constant weight)

5. Ultraviolet absorption spectrum

Only terminal absorption as shown in FIG. 1 of the accompanying drawings, when measured at 20 μg/ml in a 50% v/v aqueous methanolic solution.

6. Infrared absorption spectrum

The spectrum, measured in a KBr disc, is shown in FIG. 2 of the accompanying drawings.

7. Nuclear magnetic resonance spectrum

The spectrum, as measured in deuterochloroform as solvent and with tetramethylsilane (TMS) as internal standard, is shown in FIG. 3 of the accompanying drawings.

8. Solubility

Soluble in methanol, ethanol, ethyl acetate, acetone and chloroform.

Sparingly soluble in benzene.

Insoluble in water.

9. Colour reaction

Brown-coloured with 50% v/v aqueous sulphuric acid Positive for iodine and potassium permanganate on silica gel thin layer chromatograms. Negative for ninhydrin and 2,4-dinitrophenylhydrazine.

10. Amino acid composition

1mole each of glycine, proline, leucine, 2-amino-5-methylhexanoic acid, N-methylthreonine, N-methylleucine, methylproline and ethylproline; and 2 moles of N-methylvaline detected after hydrolysis with 6 N hydrochloric acid at 105° C. for 20 hours.

11. Antibacterial activity

Strong antibacterial activity against various bacteria of the genus Mycobacterium.

In addition, Mycoplanecin has Rf values (on silica gel thin layer chromatography, F254, 0.25 mm, No. 5715, available from Merck and Co. Inc) of 0.15 when developed with ethyl acetate and 0.64 when developed with a 95:5 by volume mixture of chloroform and methanol.

The novel microorganism of the invention is Actinoplanes nov. sp. Strain No. 41042, which was isolated from a sample of soil collected in Sumoto City, Hyogo Prefecture, Japan. The strain has been deposited under Accession No. FERM-4504 with the Technical Research Institute of the Microbial Industry, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, Japan. The strain has also been deposited under Accession No. NRRL-11462 with the U.S. Department of Agriculture.

Also provided, as part of the present invention, is a method of producing the new antibiotic substance, Mycoplanecin, which comprises cultivating a Mycoplanecin-producing microorganism of the genus Actinoplanes and then isolating Mycoplanecin from the culture.

DETAILED DESCRIPTION OF INVENTION

Morphological characteristics and physiological properties of Actinoplanes nov. sp. Strain No. 41042 are as described below:

1. Morphological characteristics

When cultivated on medium ISP4 prescribed by the International Streptomyces Project (ISP) at 28° C. for 14 days and observed under a microscope Strain No. 41042 exhibited the following morphological characteristics: spherical to subspherical sporangia with an ordinary diameter of 7-15μ; and spores of size 0.8μ×1.3μ, with flagella and having a motility in a 1/15 M phosphate buffer of pH 7.0 of about 40 minutes. The growth results on various media at 28° C. for 14 days are shown in Table 1.

                  TABLE 1                                                          ______________________________________                                         Type of              Characteristics of strain                                 medium     Item      No. 41042                                                 ______________________________________                                         Yeast . malt agar                                                                         SM        Abundant, dark yellowish orange                           (ISP 2)              (8-7-8)                                                              AM        Not grown                                                            SP        Not produced                                                         SG        Not formed                                                Oatmeal . agar                                                                            SM        Abundant, yellowish brown                                 (ISP 3)              (10-6-8)                                                             AM        Trace aerial mycelia                                                 SP        Pale yellowish brown                                                           (4-8-9)                                                              SG        Not formed                                                Starch . salts                                                                            SM        Moderate, dark yellowish                                  agar (ISP 4)         orange (8-7-8)                                                       AM        Not grown                                                            SP        Not produced                                                         SG        Abundant                                                  Glycerol . aspar-                                                                         SM        Abundant, pale yellowish                                  agine agar           brown (4-8-8)                                             (ISP 5)                                                                                   AM        Not grown                                                            SP        Not produced                                                         SG        Not formed                                                Tyrosine-  SM        Abundant, light brown                                     agar (ISP 7)         (8-6-7)                                                              AM        Not grown                                                            SP        Pale yellowish brown                                                 SG        Not formed                                                Nutrient   SM        Less good, pale orange                                    agar                 (4-8-7)                                                              AM        Not grown                                                            SP        Not produced                                                         SG        Not formed                                                Emerson's  SM        Moderate, pale yellowish                                  agar                 brown (6-8-8)                                                        AM        Not grown                                                            SP        Yellowish brown                                                                (10-6-8)                                                             SG        Not formed                                                Water      SM        Poor, yellowish gray                                      agar                 (1-9-10)                                                             AM        Not grown                                                            SP        Not produced                                                         SG        Moderately formed                                         Calcium    SM        Moderate, pale orange                                     malate               (4-8-7)                                                   agar                                                                                      AM        Not grown                                                            SP        Not produced                                                         SG        Slightly formed                                           Hickey-    SM        Abundant, yellowish brown                                 Tresner's            (8-8-8)                                                   agar       AM        Slight trace aerial mycelia                                                    grown                                                                SP        Not produced                                                         SG        Not formed                                                Bennett's  SM        Abundant, dark yellowish                                  agar                 orange (8-7-8)                                                       AM        Not grown                                                            SP        Not produced                                                         SG        Slightly formed                                           Sucrose-   SM        Less good, pale orange                                    nitrate              (4-8-7)                                                   agar                                                                                      AM        Not grown                                                            SP        Not produced                                                         SG        Abundant                                                  Glucose .  SM        Moderate, brownish white                                  asparagine           (2-9-8)                                                   agar                                                                                      AM        Not grown                                                            SP        Not produced                                                         SG        Slightly formed                                           Tomato paste .                                                                            SM        Moderate, pale yellowish brown                            oatmeal              (6-8-9)                                                   agar                                                                                      AM        Not grown                                                            SP        Not produced                                                         SG        Not formed                                                Glycerol . SM        Abundant, pale yellowish brown                            glycin               (6-8-8)                                                   agar                                                                                      AM        Not grown                                                            SP        Not produced                                                         SG        Not formed                                                Glucose .  SM        Moderate, dark yellowish                                  nitrate              orange (8-7-8)                                            agar                                                                                      AM        Not grown                                                            SP        Dark yellow (10-7-9)                                                 SG        Not formed                                                ______________________________________                                          Abbreviations:                                                                 SM: Substrate mycelium                                                         AM: Aerial mycelium                                                            SP: Soluble pigment                                                            SG: Sporangium                                                           

Physiological characteristics

The physiological characteristics of Strain No. 41042 are given in Table 2.

                  TABLE 2                                                          ______________________________________                                         Tyrosinase reaction                                                                               Positive (strong)                                           Nitrate reduction  Positive (strong)                                           Hydrolysis of starch                                                                              Negative                                                    Gelatin liquefaction                                                                              Positive (strong)                                           Peptonization of milk                                                          26° C.      Positive (pH 6.0)                                           Coagulation of milk                                                            26° C.      Positive                                                    Melanin formation:                                                             Medium A           Positive                                                    Medium B           Positive                                                    Temperature range for                                                          growth             9.0-32.5° C.                                         (on ISP 2 medium)  (Optimum temperature                                                           for growth 18.5-29° C.                               ______________________________________                                    

The media used were as follows:

Medium A: Tryptone--yeast extract broth (ISP 1).

Medium B: Peptone--yeast extract--iron agar (ISP 5).

The carbon source utilization pattern of Strain No. 41042 on Pridham-Gottlieb's agar medium at 28° C. after 14 days is shown in Table 3.

                  TABLE 3                                                          ______________________________________                                         Medium    C      D       Medium      C    D                                    ______________________________________                                         D-Glucose ++     ++      Raffinose   -    -                                    L-Arabinose                                                                              ++     ++      Inulin      -    -                                    D-Xylose  ++     ++      Dextrin     ++   ++                                   D-Fructose                                                                               +      ++      Starch      +    +                                    L-Rhamnose                                                                               +      ++      i-Inositol  -    -                                    D-Galactose                                                                              ++     ++      D-Mannitol  -    +                                    D-Mannose ++     ++      Dulcitol    -    -                                    Sucrose   +      ++      Salicin     -    -                                    D-Cellobiose                                                                             +      ++      Sodium acetate                                                                             -    -                                    Melibiose -      -       Sodium succinate                                                                           -    -                                    β-Lactose                                                                           -      +       Glycerol    -    ++                                   Maltose   +      +       Cellulose   -    -                                    Trehalose -      +       Control     -    -                                    ______________________________________                                          ++: Well utilized                                                              +: Utilized                                                                    -: Not utilized                                                          

The following media were used:

Medium C: Pridham-Gottlieb's agar.

Medium D: Pridham-Gottlieb's agar plus yeast extract 0.5%.

3. Mycelium components

An acid hydrolyzate of the mycelia was analyzed by paper chromatography according to the methods described by B. Becker et al in Applied Microbiology, 12, 421-423 (1964) and 13, 236-243 (1965) and by M. P. Lechevalier et al in "The Actinomycetales" by H. Prauser, 311-316 (1970). The results are shown in Tables 4 and 5.

                  TABLE 4                                                          ______________________________________                                         Cell wall type                                                                 Type of diaminopimelic                                                         acid           Glycine  Arabinose  Galactose                                   ______________________________________                                         Meso  LL      Hydroxy                                                          +     -       +        +      Trace    +                                       ______________________________________                                    

From these results, it is confirmed that the cell wall type is II.

                  TABLE 5                                                          ______________________________________                                         Saccharide components in whole cell                                            ______________________________________                                         Glucose Galactose  Mannose   Arabinose                                                                               Xylose                                   +       +          Trace     Trace    Trace                                    ______________________________________                                    

As can clearly be seen from the properties described above, Strain No. 41042 belongs to the genus Actinoplanes of the family Actinoplanaceae, within the group of actinomycetes. This conclusion is based upon the formation of spherical to subspherical sporangia, motile spores and cell walls of type II; the new strain is therefore named Actinoplanes nov. sp. Strain No. 41042.

As is well-known, the properties of actinomycetes including Actinoplanes, strains are not fixed and they readily undergo mutation both through natural causes and as the result of artificial mutation. Although the invention relates to the production of the new antibiotic Mycoplanecin especially by the cultivation of the above-defined Actinoplanes nov. sp. Strain No. 41042, it also includes within its scope the use of mutants of this organism and generally of any Actinoplanes strain which is capable of producing Mycoplanecin, in order to obtain this antibiotic substance.

The cultivation of the Mycoplanecin-producing microorganism, in accordance with the process of the invention, can be performed under the conditions conventionally employed for the cultivation of Actinoplanes species. Shaken culture or submerged culture with aeration and agitation in a liquid medium are preferred.

The nutrient medium used for the cultivation can be of a composition such as is conventionally used for the cultivation of actinomycetes. Thus, it contains: an assimilable carbon source, e.g. glucose, arabinose, galactose, mannose, sucrose, maltose, dextrin, starch, glycerin, a vegetable fat or oil (such as soybean oil, corn oil or cottenseed oil), an animal fat or oil (such as chicken oil or lard) or fish oil, an assimilable nitrogen source, e.g. soybean meal, peanut oil, cottonseed meal, fish meal, corn steep liquor, oatmeal, skimmed milk, peptone, meat extract, live yeast, yeast extract, casamino acid, sodium nitrate, ammonium nitrate or ammonium sulphate; and an inorganic salt, e.g. sodium chloride, potassium chloride, phosphates, magnesium carbonate, calcium carbonate or calcium chloride. It may also contain minor amounts of various other metal salts, for example ferrous sulphate, copper sulphate, magnesium sulphate, zinc sulphate or cobalt sulphate.

Cultivation in a liquid medium is preferably performed aerobically, with aeration and agitation, in which case an anti-foaming agent may be added to the medium: suitable anti-foaming agents include silicone oils, vegetable oils or surfactants.

The cultivation is suitably performed at a substantially neutral pH value and at a temperature of from 18° to 30° C., preferably about 28° C.

The titre of Mycoplanecin produced in the culture broth as cultivation proceeds can be quantitatively determined by the cup assay method using Mycobacterium smegmatis ATCC 607 as the test microbe. The maximum production of Mycoplanecin is generally achieved after 3-5 days of cultivation.

Mycoplanecin is present in both the liquid portion and the mycelial portion of the culture broth produced by the process of the invention. In order to recover the antibiotic from the culture broth on completion of the cultivation, the mycelium and other solids are first removed from the liquid phase by filtration, for example using diatomaceous earth as a filter aid, or by centrifugation. The Mycoplanecin which is present in the mycelial portion on the filtrate or the supernatant can then be isolated and purified by conventional techniques suited to its physico-chemical properties.

For example, the Mycoplanecin in the mycelial portion can be extracted by the addition of a water-miscible solvent, such as methanol, ethanol, isopropanol or acetone. The solvent is then removed from the extract and the residue is re-extracted with a water-immiscible solvent, such as chloroform, ethyl acetate, methyl isobutyl ketone or methylene chloride. The Mycoplanecin contained in the liquid portion of the culture broth can also be extracted with such a water-immiscible solvent, after which it would normally be combined with the extract from the mycelial portion and concentrated to give a crude Mycoplanecin extract.

Alternatively, Mycoplanecin can be extracted by adding a water-miscible solvent, such as those exemplified above, directly to the culture broth without separating the mycelium from the liquid portion; the resulting extract is filtered and concentrated to remove the solvent and then the residue is re-extracted, as in the procedure described above, with a water-immiscible solvent.

The Mycoplanecin thus obtained may be further purified by any of the methods well-known for the purification of compounds having similar physico-chemical properties; however, we prefer to use purification techniques employing an adsorbent or a counter-current distribution method. Suitable adsorbents include alumina, silica gel, Sephadex (a Trade Mark for a range of polysaccharide-derived organic compounds) or cellulose. We particularly prefer a column chromatography separation method employing silica gel as carrier and methanol, ethyl acetate or chloroform or any mixture thereof as eluent. Preparative thin layer chromatography employing silica gel is also effective for producing more highly purified Mycoplanecin.

The purified Mycoplanecin thus obtained shows single spots in coloration reactions with sulphuric acid, potassium permanganate or iodine on a silica gel thin layer chromatogram and also has the physical and chemical properties heretofor described.

The minimal inhibitory concentrations (MIC) of Mycoplanecin against various microorganisms is shown in Table 6. The evaluation was made as follows: for mycobacteria, after incubation for 5 weeks at 37° C. by a dilution method employing a 1% bovine serum-containing Dubos liquid medium; on general bacteria, after incubation for 24 hours at 37° C. by an agar dilution method, employing a heart infusion agar medium; and on fungi and yeasts, after incubation for 48 hours at 26° C. by an agar dilution method employing Sabouraud's agar medium.

                  TABLE 6                                                          ______________________________________                                                                         MIC                                            Test organism          Medium   (μg/ml)                                     ______________________________________                                         Mycobacterium kansasii                                                                         IFM 2069   DM       0.025                                      Mycobacterium fortuitum                                                                        IFM 2079   DM       1.56                                       Mycobacterium intracellulare                                                                   IFM 2073   DM       0.39                                       Mycobacterium intracellulate                                                                   IFM 2083   DM       0.39                                       Mycobacterium tuberculosis                                                                     IFM 2026   DM       0.135                                      Matsodo                                                                        Mycobacterium tuberculosis                                                                     IFM 2027   DM       0.39                                       Maru                                                                           Mycobacterium tuberculosis                                                                     IFM 2028   DM       0.78                                       H.sub.37                                                                       Mycobacterium tuberculosis                                                                     IFM 2029   DM       0.39                                       H.sub.37 R.sub.v                                                               Mycobacterium tuberculosis                                                                     IFM 2030   DM       0.78                                       H.sub.2                                                                        Mycobacterium bovis BCC                                                                        IFM 2031   DM       0.78                                       Mycobacterium bovis ushi-10                                                                    IFM 2032   DM       0.1                                        Mycobacterium tuberculosis                                                     (Streptomycin-resistant)                                                                       IFM 2033   DM       0.1                                        Mycobacterium smegmatis                                                                        ATCC 607   DM       0.08                                       Mycobacterium phlei        DM       0.01                                       Micrococcus luteus                                                                             PCI 1001   H        0.0125                                     Staphylococcus aureus                                                                          209P 3C-1  H        >400                                       Bacillus subtilis                                                                              PCI 219    H        >400                                       Escherichia coli                                                                               NOHO OC-2  H        >400                                       Klebsiella pneumoniae                                                                          PCI 602    H        >400                                       Proteus vulgaris                                                                               OX 19      H        >400                                       Proteus mirabilis                                                                              1331       H        >400                                       Pseudomonas aeruginosa                                                                         SANK                                                                           73860      H        >400                                       Candida albicans                                                                               Yu 1200    S        >400                                       Aspergillus oryzae                                                                             SANK                                                                           11262      S        >400                                       Penicillium chrysogenum                                                                        SANK                                                                           12768      S        >400                                       Trichophyton mentagrophytes                                                                    SANK                                                                           22374      S        >400                                       Pyricularia oryzae                                                                             SANK                                                                           16975      S        >400                                       ______________________________________                                          *Medium DM: Dubos medium, except that 10% of bovine serum was added other      than for the two strains Mycobacterium smegmatis ATCC 607 and                  Mycobacterium phlei.                                                           Medium H: Heart infusion agar medium.                                          Medium S: Sabouraud's agar medium.                                       

The toxicity of the antibiotic substance, Mycoplanecin, was investigated by administering it intraperitoneally to mice at a dose of 100 mg/kg body weight; no mice died.

The preparation of Mycoplanecin is illustrated by the following non-limiting Examples.

EXAMPLE 1

To a 500 ml Sakaguchi flash were added 100 ml of a seed culture medium having a pH of 7.0 before sterilization and the following composition (percentages are w/v):

    ______________________________________                                         Glucose                 1%                                                     Glycerine               1%                                                     Oatmeal                 0.5%                                                   Sucrose                 1%                                                     Soybean meal            2%                                                     Casamino acid           0.5%                                                   Live yeast              1%                                                     Calcium carbonate       0.1%                                                   Water q.s.                                                                     ______________________________________                                    

This medium was inoculated with a culture of Actinoplanes nov. sp. Strain No. 41042 and reciprocal shaking culture was carried out at 28° C. for 96 hours. The resulting culture broth was divided into 5 ml portions and each portion was inoculated into a Sakaguchi flask each flask containing 100 ml of a production medium having a pH of 7.0 before sterilization and having the following composition (percentages are w/v):

    ______________________________________                                         Glycerine              0.5%                                                    Sucrose                2%                                                      Soybean meal           1%                                                      Live yeast             1%                                                      Corn steep                                                                     liquor                 0.5%                                                    COCl.sub.2 . 6H.sub.2 O                                                                               0.001%                                                  Water q.s.                                                                     ______________________________________                                    

Reciprocal shaking culture was then carried out at 28° C. for 96 hours. The resulting culture broths were combined.

To 4 liters of the combined culture broths (pH 7.2) were added 5% w/v of Celite 545 (a Trade Mark for a filter aid available from Johns Manville Product Corporation, U.S.A.) and the broth was filtered to separate the liquor from the mycelia-containing filter cake. The filtrate was extracted with 2 liters of ethyl acetate to recover its Mycoplanecin content, whilst the mycelial cake was extracted with 2 liters of acetone containing 20% v/v water; the acetone was distilled off under reduced pressure and the residue was extracted with 2 liters of ethyl acetate. The ethyl acetate extracts from the filtrate and the mycelial cake were combined, to give 4 liters of combined extracts.

These combined extracts were washed twice, each time with 1 liter of a saturated aqueous solution of sodium chloride. The washed extracts were dehydrated over anhydrous sodium sulphate and then concentrated by evaporation under reduced pressure to give 1.07 g of an oily substance.

This oily substance was dissolved in a small volume of chloroform and adsorbed on a column containing 20 g of silica gel which had previously been prepared with chloroform. The column was then washed with chloroform and impurities were eluted away using a 1:1 by volume mixture of chloroform and ethyl acetate and then ethyl acetate alone. The desired Mycoplanecin was then eluted with a mixed solvent containing 95% by volume ethyl acetate and 5% by volume methanol. One liter of an active fraction was separated from the eluted fractions and concentrated by evaporation under reduced pressure to give 130 mg of a white powder.

110 mg of this white powder were dissolved in a small volume of chloroform and the resulting solution was passed into a 200 ml column containing Sephadex LH-20 (available from Pharmacia Co. Limited, Sweden) packed with chloroform, and which was then eluted with chloroform. Active fractions thus collected were concentrated by evaporation under reduced pressure to give 90 mg of Mycoplanecin as a white powder. This purified product showed single spots with iodine, sulphuric acid and potassium permanganate on a silica gel thin layer chromatograph.

EXAMPLE 2

Ten 500 ml Sakaguchi flasks, each containing 100 ml of a seed culture medium having the same composition as that described in Example 1, were inoculated with a culture of Actinoplanes nov. sp. Strain No. 41042, and reciprocal shaking culture was conducted at 28° C. for 96 hours.

A 30 liter jar fermenter, which contained 15 liters of a production medium having the same composition as that described in Example 1, was inoculated with 750 ml of the culture broth produced as described above and then this was subjected to shaking culture with agitation and aeration at 250 rpm, a temperature of 28° C. and an aeration of 15 liters/minute for 80 hours.

To the resulting culture broth were added 15 liters of acetone, the mixture was stirred and the product was filtered with the aid of 500 g of Celite. 25 liters of the filtrate were concentrated by evaporation under reduced pressure to distill off the acetone. To the residual liquid (15 liters) were added 7 liters of methylene chloride and extraction was carried out with shaking and agitation. The residue was further extracted with another 7 liters of methylene chloride and the extracts were combined, after which the solvent was evaporated off to give 1.8 g of an oily substance.

A solution of this oily substance in a small volume of chloroform was adsorbed on a column containing 50 g silica gel and packed with chloroform, from which Mycoplanecin was eluted with a solvent system comprising 99% by volume chloroform and 1% by volume methanol.

400 ml of active fraction were collected and concentrated by evaporation under reduced pressure to give 390 mg of a white powder. A solution of this white powder in a small volume of chloroform was then subjected to preparative thin layer chromatography employing a silica gel plate (5717, available from Merck and Co. Inc.) developed with a 95:5 by volume mixture of chloroform and methanol. The active bands were extracted with ethyl acetate to give 250 mg of Mycoplanecin as a white powder having a purity of about 90% (by bioassay). 

We claim:
 1. A biologically pure culture of Actinoplanes nov. sp. Strain No. 41042, NRRL-11462, said culture being capable of producing Mycoplanecin. 